Transport peptides such as C-terminal Erns peptide and analogues thereof

ABSTRACT

The invention relates to peptides derived from or similar to the E rns  protein of pestiviruses for type-specific diagnosis of infection, for eliciting antibiotic activity, and for transport of substances into a cell. For these purposes, the invention provides, among other things, an isolated, synthetic or recombinant protein or peptide module or functional equivalent thereof comprising an amino acid sequence having significant homology to, for example, an amino acid sequence of a peptide located from about amino acid position (a) 194 to about 220 in a pestiviral E rns  protein (b) 59 to about 88 in an L3 loop of a cytotoxic RNase of a ribosome-inactivating protein, or (c) 187 to about 223 in a respiratory syncytial virus G-protein.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of application Ser. No.10/335,057, filed Dec. 30, 2002 now U.S. Pat. No. 7,067,487, which is acontinuation of International Application No. PCT/NL01/00484, filed Jun.28, 2001, designating the United States of America, and published inEnglish as WO 02/00882 A2 on Jan. 3, 2002, the contents of the entiretyof which is incorporated herein by this reference.

TECHNICAL FIELD

The various embodiments of the present invention generally relate tobiotechnology. More specifically, embodiments of the invention relate totransport peptides, for example, derived from the E^(rns) protein ofpestiviruses for type-specific diagnosis of infection, for elicitingantibiotic activity and for transport of substances into a cell.

BACKGROUND

Hog cholera virus or classical swine fever virus (CSFV), bovine viraldiarrhea virus (BVDV), and border disease virus (BDV) belong to thegenus Pestivirus of the Flaviviridae family. CSFV is restricted toswine, while BVDV and BDV have been isolated from several species suchas cattle, swine, sheep, deer, and giraffes. Although pigs can beinfected by all of these pestiviruses, only CSFV induces severe, oftenfatal, disease. The disease is characterized by fever and, for instance,leukopenia and can run an acute, chronic, or subclinical course.Although effective live-attenuated vaccines are available, pigs are notvaccinated against CSFV in the European Union (EU) because vaccinatedand infected pigs are serologically indistinguishable. Outbreaks of CSFin the EU are controlled by eradication of all pigs from infected farmsand farms in the vicinity. Because of this strategy, more than 10million pigs had to be killed and destroyed during the 1997-1998 CSFepizootic in The Netherlands, costing more than $2 billion. It is forthis reason that a great demand exists for a marker vaccine thatprovides protective immunity and induces an antibody response in thevaccinated pigs that can be distinguished from the antibody responsecaused by a natural CSFV infection.

Like other members of the family, pestiviruses are plus-stranded RNAviruses whose genome comprises one long open reading frame. Translationinto a hypothetical polyprotein is accompanied by processing into matureproteins. The structural proteins include a nucleocapsid protein C andthree envelope glycoproteins E^(rns), E1 and E2. The envelope proteinsE^(rns) and E2 are able to induce neutralizing antibodies. GlycoproteinE2 is the most immunogenic protein of pestiviruses and elicits hightiters of neutralizing antibodies after infection. Vaccination of targetanimals with E2 has shown to give complete protection against a lethalhomologous challenge. When E2 is used for vaccination, serologicaldiagnosis of a natural pestivirus infection has to be performed with animmunogenic/antigenic protein other than E2 that is present in theinfectious pestivirus. For this purpose, the E^(rns) glycoprotein can beused as an antigen in a diagnostic test. A population that is vaccinatedwith the E2 glycoprotein can still be tested serologically forpestivirus infection with a diagnostic test based on the E^(rns)antigen. A serological test based on E^(rns) can distinguish E^(rns)antibody-positive sera from animals infected with the virus and E^(rns)antibody-negative sera from uninfected animals. This is called themarker vaccine approach. Of course, these marker vaccines depend onsensitive tests and, in the case of CSFV, the test also has to be veryspecific because pigs can be infected with the other pestiviruses BVDVand BDV. Because BVDV and BDV do not cause (severe) clinical symptoms inpigs and the animals are not vaccinated for these viruses, thediagnostic test for a CSFV marker vaccine should only detect CSFVantibodies and no other pestivirus antibodies.

Serological tests based on the complete E^(rns) protein have beendeveloped previously but are not always satisfactory in that they arenot specific enough in that they cannot discriminate sufficientlybetween infections with different pestivirus species or are notsensitive enough to detect early infections with a pestivirus.

In one embodiment, the invention provides a so-called transport peptidemodule, herein also called “movin.” In principle, we found that mostlinear peptides of 10 to 18 residues long which have >40% arginines (R)or lysines (K) are capable of functioning as such a transport peptidemodule to which cargo can be attached. Such a transport peptide modulepreferably should not, or only to a small extent, contain negativelycharged amino acids such as aspartic acid (D) or glutamic acid (E).Preferred peptide modules are identified herein with full sequence, suchas, for example, in Tables 4, 5, and 9 to 11, or retro-inverso variantsthereof.

Variations in amino acid sequence are well tolerated, at least from theviewpoint of translocation as activity. As a rule of thumb, it can besaid that related sequences have at least 30-50% homology, preferably atleast 70% homology, and most preferably at least 85% homology, to thosedisplayed in these tables, which allows identifying further relevantsequences present in nature or capable of being synthesized.

Substitutions in the amino acid sequence of a transport peptide modulecan be applied to increase the translocation (transport) activity. Anoptimized transport peptide module can, for example, be synthesizedaccording to retro-inverso peptide chemistry, in which the sequence isreversed and D-amino acids are used instead of L-amino acids. Transportpeptides derived from the herein-indicated positions of the E^(rns)peptide, L3 loop peptides or human respiratory syncytial virus protein G(HRSV-G) peptides, and peptide mimics or peptoides derived thereof wereable to bind surface glycosaminoglycans like heparin. Therefore, findingthat a peptide belongs to the group of linear heparin-binding peptidesor is capable of binding related glycosaminoglycans can be used as aprediction that they likely also can function as transport peptides.However, heparin binding is not a prerequisite for a peptide being atransport peptide.

To check if the presence of heparin on the surface of the cellinfluenced the efficiency of translocation, it was tested whetherheparin-binding peptides also translocated into mutant cells which wereglycosaminoglycan deficient (cell lines pgsA-745 and pgsD-677).Titrations of all heparin-binding peptides on the different cells showedthat peptides translocated with the same efficiency/activity intoheparin-containing cells and in the mutant cells without heparin (datanot shown). Thus, heparin-binding peptides have translocation activity,and binding of the peptides to heparin obviously does not block thepeptide from penetrating the plasma membrane. Likely, the peptides havea high on/off rate for heparin, and the high affinity for phospholipidsdirects the peptides to the membrane and ultimately into the cell. Onthe other hand, heparin binding, albeit being predictive, does not seemto be a prerequisite for efficient translocation of the peptides.

The invention further provides a method for translocating a compoundover a membrane of a cell, an epithelial layer, mucus layer, blood-brainbarrier or skin comprising providing the compound with a transportpeptide module according to the invention and contacting it with a cell.Such compounds, herein also called cargo, can be large; successfultranslocation of compounds up to 600 kD has been demonstrated and it isexpected that even larger compounds may be translocated. From theperspective of speed of translocation in relation to the usefulness ofthe compound, compounds of preferred molecular weight are those of 60 to500 kD and even more preferred are those of 120 to 300 kD. Compounds canalso be of a varied nature. For example, it is possible to linkmacromolecules such as nucleotides, polypeptides, drugs such asantiviral, antimicrobial or anti-inflammatory drugs, and the like to amodule as provided herein for successful translocation of such acompound. Topical application of such a compound, e.g., as apharmaceutical composition, is specifically provided. A module asprovided herein has excellent capacity to penetrate to the upper layersof the skin. Typical applications include further use of a labile linkersuch as a thioester or a O(C═O) CH₂NRC (═O) CH₂NHCH₂ (C═O) SCys linker.For use of a transport peptide module according to the invention, drugsor macromolecules are typically covalently coupled to the peptide,examples of which are cyclosporine A, acyclovir, and terbenafine coupledwith a module according to the invention.

This invention also provides, among others, peptide-based diagnostics inconnection with diseases caused by pestivirus infections. Antigenicpeptides as provided herein and useful for diagnostics can surprisinglyalso be used otherwise, such as antibacterial or transport peptides.Because in one embodiment the transport peptide module is a fragmentderived of the E^(rns) protein, it can be used for diagnosis ofpestivirus infections when a marker vaccine is used that is based on E2,another pestivirus surface protein. Due to its unique biochemicalcharacter, a peptide as provided herein has the ability to permeate andkill microorganisms and has the ability to translocate itself and acoupled cargo across a cell membrane and epithelium barrier.

In a preferred embodiment, the invention provides a thus farunidentified small, independently folding protein (peptide) modulerelated to modules present at the C-terminal end of pestivirus E^(rns),at the L3 loop of secreted cytotoxic Rnases that preferably belong tothe group of type II ribotoxins such as alpha-sarcin, restrictocin,mitogillin, toxin Asp fi, clavin or gigantin, in a heparin-bindingpeptide, in a DNA/RNA-binding peptide, in HRSV-G protein, and its use asa transport peptide. Previously, the region responsible fortranslocation of alpha-sarcin was thought to be located in a hydrophobicstretch, located away from the L3 loop (Mancheno et al., Biophys. J. 68,2387-2395, 1995). In a preferred embodiment, the invention provides anisolated, synthetic or recombinant protein module or functionalequivalent thereof comprising an amino acid sequence that is at least85% identical to any of the sequences shown in Tables 1-4 and 9-11,e.g., to an amino acid sequence of a peptide located from about aminoacid position 194 to 220 in a pestiviral E^(rns) protein and/or that isat least 70% identical to an L3 loop sequence such as shown in Table 5.

Such transport peptide modules can be prepared synthetically with normalpeptide synthesis or coupling techniques as described herein, startingfrom individual amino acids or by coupling or linking smaller peptidesof relevant sequence to another or by cleaving off from larger peptides.When desired, nonconventional amino acids can be used, such as D-aminoacids or others that normally do not occur in natural proteins. Peptidescan also be prepared via recombinant DNA techniques via transcriptionand translation from recombinant nucleic acid encoding such a peptide orprotein module, be it linked to, for example, a fusion protein orspecific target molecule such as a desired binding molecule derived froman antibody or protein ligand or receptor-binding molecule, and so on.For example, we have successfully expressed a fusion protein of atransport peptide and Green Fluorescent protein in A72 cells. The GreenFluorescent protein showed the same cellular localization as thebiotinylated transport peptide in the nucleoli and around the nucleus.This is in contrast to normally expressed Green Fluorescent protein,which was distributed evenly over the cell (data not shown).

In a preferred embodiment, the invention provides a transport peptidemodule or functional part thereof wherein at least the functional partof the peptide comprises a reversed amino acid sequence to one of asequence given in claims 1 to 6 and wherein D-amino acids are usedinstead of L-amino acids. Reversing the sequence and using the D-aminoacids instead enhances translocation activity, allowing improved usefor, for example, transport of macromolecules or drugs through cellmembrane barriers into cells.

In a preferred embodiment as explained herein, the invention provides amodule which is functional as a transport peptide module, also whencargo is attached, wherein the peptide is located from about amino acidposition 191 to 222, or from about 194 to 227, or from about 191 to 227,or from about amino acid position 176 to about 220, 222, or 227 in thecase of the pestiviral E^(rns) protein or residues 51-91 or 59-88 orfrom 62-88 or from 62-74, in the case of the L3 loop protein, or fromabout amino acid position 187 to 223 in a respiratory syncytial virusG-protein. Also, in HRSV type B, a similar region was detected fromposition 149 to 160 in protein G. These amino acid positions and theirnumbering are, of course, relative to known sequences as, for example,shown in the figures herein wherein alignments of various pestiviralsequences are shown, which, of course, allows, for example, foralignment with yet unknown pestiviral sequences and allows alignmentwith ribotoxin L3 loop sequences. As a rule of thumb, it can be saidthat related sequences have at least 30-50% homology, preferably atleast 70% homology, most preferably at least 85% homology, which allowsidentifying further relevant sequences present in nature or capable ofbeing synthesized. As examples herein, modules are described wherein thepeptide comprises the amino acid sequence RQGAARVTSWLGRQLRIAGKRLEGRSK(SEQ ID NO:1); RQGTAKLTTWLGKQLGILGKKLENKSK (SEQ ID NO:2);RVGTAKLTTWLGKQLGILGKKLENKTK (SEQ ID NO:3); RQGAAKLTSWLGKQLGIMGKKLEHKSK(SEQ ID NO:4); GNGKLIKGRTPIKFGKADCDRPPKHSQNGMGK (SEQ ID NO:5);GDGKLIPGRTPIKFGKSDCDRPPKHSKDGNGK (SEQ ID NO:6);GEGKILKGRTPIKFGKSDCDRPPKHSKDGNGK (SEQ ID NO:7);GDGKILKGRTPIKWGNSDCDRPPKHSKNGDGK (SEQ ID NO:8); KRIPNKKPGKK (SEQ IDNO:9); KTIPSNKPKKK (SEQ ID NO:10); KPRSKNPPKKPK (SEQ ID NO:11); or afunctional part thereof. However, variations can be introduced, forexample, by increasing the positive charge of the peptide, preferably atpositions that optimize the amphipathic nature of the peptide, but notnecessarily. Another example is changing several or all L-amino acids toD-amino acids to reduce possible protease sensitivity. The translocationactivity of the E^(rns) peptide was further improved by substitution ofthe 2 lysines and the glutaminic acid by arginines. In a preferredembodiment, a retro-inverso variant of an above-identified peptidemodule is provided; such a retro-inverso peptide with an inversedsequence and D-amino acids replacing L-amino acids comprises even highertranslocation activity.

Of course, the invention also provides a recombinant nucleic acidencoding a module according to the invention, for example, to providefor a proteinaceous substance provided with a module according to theinvention, for example, provided with a targeting means.

The invention in one aspect also relates to the design of an antigenicsubstance, preferably peptide-based, corresponding to the protein modulein the E^(rns) protein of Pestiviruses or a L3 loop of ribotoxin II canbe used as a basis for, e.g., diagnostics tests, antibacterial ortransporter peptides. For example, in one embodiment, the inventionprovides a method for inducing an antibody comprising administering amodule or a substance according to the invention to a host capable offorming antibodies. Antibodies can be induced classically by, forexample, immunizing an animal with the antigenic substance, or via moremodern techniques, such as phage display, whereby so-called syntheticantibodies are produced. Be it synthetic or classical (mono- orpolyclonal), the invention provides an antibody specifically directedagainst a module according to the invention.

With the pestivirus-derived module and/or the antibody as providedherein, the invention provides a method for detecting the presence orabsence of an antibody directed against a pestivirus in a samplecomprising contacting the sample with a module or a substance accordingto the invention, the method preferably further comprising detecting thepresence or absence of an antibody bound to the module or substance.Also provided is a method further comprising contacting the sample withthe module or substance in the presence of a competing antibody directedagainst the module and detecting the presence or absence of competingantibody bound to the module or substance. Herewith, the inventionprovides use of a method according to the invention for differentiatingat least one animal from at least another animal. The invention thusprovides a test which is based on a small fragment of the E^(rns)protein. Sequence analysis and homology modeling was used for pestivirusE^(rns) to identify a region that can be used for the design ofantigenic substances and resulted in the identification of a smallindependently folding protein module which, in its native state, isexposed on the protein surface of the complete E^(rns) protein and canbe used to design antigenic substances which are comparable or superiorto the complete protein.

In a further embodiment, the invention not only provides a peptide thatbehaves as a superior antigen in the E^(rns) peptide-ELISA but one thathas additional characteristics that are very interesting and useful. Dueto its unique biochemical nature, a peptide as provided herein, forexample, corresponding to the E^(rns) C-terminal domain or to a L3 loopin a ribotoxin, is able to interact with a cell membrane and destabilizethe membrane.

The invention further provides a method for translocating a compoundover a membrane of a cell, an epithelial layer, mucus layer, blood-brainbarrier or skin comprising providing the compound with a module orsubstance or transport peptide module according to the invention andcontacting it with a cell, and, furthermore, it provides a method foreliciting antibiotic activity to a microorganism comprising contactingthe microorganism with the module or substance.

Herein, it is shown that such an E^(rns) peptide or protein module asprovided herein has antibacterial activity for, for example,gram-negative bacteria (E. Coli) and an L3 loop or E^(rns) peptide andit has translocation activity for, for example, eukaryotic cellmembranes. A biological membrane is a very efficient barrier thatprotects the micromilieu of cells or intracellular compartments from theoutside milieu. In order to interfere directly with biological processesinside the cell, it is necessary that pharmaceuticals cross the lipidbilayer to block/bind their targets. Many promising, potentialtherapeutics (hydrophilic organic molecules, peptides, proteins orgenes) are ineffective because the cell membrane forms an insurmountablebarrier. However, several peptides have been discovered recently thatcan solve this problem because they are able to translocate over thelipid bilayer and are also able to transport a diverse set of cargosinside the cell.

Interactions of pore-forming peptides with model and artificialmembranes have been studied extensively the last three decades. Severalfamilies of membrane destabilizing peptides with antitumor, haemolytic,antibacterial activity or a combination have been found. Many of thesepeptides form amphipathic helices with a hydrophobic face and a positivecharged face that organize and aggregate on the membrane surface anddestabilize the membrane. Their mode of action has some resemblance withthe recently discovered transport peptides (Matsuzaki et al., Biochem.Biophys. Acta. 1376: 391-400, 1998; Lindgren et al., Trends Pharmacol.SCI 21: 99-103, 2000). The invention now provides a pharmaceuticalcomposition comprising a module or substance according to the inventionuseful for several purposes. For example, the invention provides use ofa module or a substance according to the invention for the preparationof a pharmaceutical composition capable of membrane translocation (atransport peptide), for the preparation of a pharmaceutical compositioncapable of eliciting antibiotic activity (an antibiotic), or for thepreparation of a pharmaceutical composition capable of inducingantibodies (a vaccine) upon administration to a host.

The invention is further explained in the detailed description describedherein without limiting it thereto.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

FIG. 1. Schematic representation of alignment of pestivirus E^(rns) withRNase Rh which indicates the modular organization of E^(rns). E^(rns)consists of an RNase domain (dotted) and a C-terminal membrane-activedomain (filled black). The C-terminal domain (residues 191-227) whichshows resemblance to the L3 loop of cytotoxic RNases is described inthis invention. RNase-active site domains are shown as checkered boxes.Potential glycosylation sites are shown as ellipses.

FIG. 2. Helical wheel representation of residues 194-220 of CSFVE^(rns).

FIG. 3. Sequence alignment of pestivirus E^(rns) C-terminal domains withmagainin and the L3 loop of restrictocin. Residues within one distanceunit from magainin are boxed. Units are defined in structural distancetable in the magalign package of DNASTAR, Inc. The Structural tablescores for residues that are chemically and spatially similar. Allidentities score a value of 6. Mismatches score less than identities.The Structural table is designed for use with the Jotun-Hein method.

FIG. 4. Crystal structure of RNAse Rh.

FIGS. 5A and 5B. Reactivity (optical density, OD) in CSFV 1p-peptideELISA of dilutions of BVDV-specific swine sera (4b-9b) and CSFV-specificswine sera (9c to 13c) and CSFV-specific hyperimmune serum.

FIGS. 5C and 5D Reactivity in CSFV 1p-peptide ELISA of dilutions ofBVDV-specific bovine sera (1-6, r4590-51, r4590-52, 841) andBVDV-specific hyperimmune serum.

FIGS. 6A and 6B. Reactivity of several panels of sera in the CSFV1p-peptide ELISA as described in the test procedure. Negative fieldserum samples (n=96) were randomly obtained from slaughtered adult pigsand were all tested negative in standard pestivirus ELISA.Pestivirus-positive but CSFV-negative serum samples (n=96) were randomlyobtained from slaughtered adult pigs. CSFV-positive field serum sampleswere obtained (n=95) from an infected farm (VR) that was infected duringthe CSF epizootic in the Netherlands in 1997-1998.

FIG. 7. Reactivity of successive serum samples collected during avaccination/challenge experiment in the CSFV 1p-peptide ELISA. Twelvepigs were vaccinated with E2, 14 days before challenge.

FIGS. 8A-8C. Distribution of biotinylated CSFV E^(rns) peptide (8A, 8C)and biotinylated control peptide (8B) (25 μM) after 30 minutes ofincubation with subconfluent EBTr cells grown on a 10-well microscopeslide. Cells were fixed with cold methanol and biotinylated peptide wasvisualized by staining with avidin-FITC for 30 minutes. Fluorescentmicrograph (250×) (8A, 8B) or fluorescent micrograph using confocalmicroscope (600×) (8C).

FIGS. 9A and 9B. Distribution of biotinylated L3 peptide (9A) andmagainin-1 peptide (9B) (6 μM) after 30 minutes of incubation withsubconfluent EBTr cells grown on a 10-well microscope slide. Cells werefixed with cold methanol and biotinylated peptide was visualized bystaining with avidin-FITC for 30 minutes. Fluorescent micrograph (250×).

FIGS. 10A-10H. Transport of avidin and streptavidin. Equimolar amountsof avidin-Texas Red (66 kD) (10A) or streptavidin-FITC (60 kD) (10C)were mixed with the biotinylated E^(rns) peptide or unbiotinylatedpeptide (10B, 10D) (residues 194-220) (3 μM) and incubated with EBTrcells for 30 minutes. Transport of a complex of streptavidin-FITC withoptimized E^(rns) peptide biotin-GRQLRIAGRRLRGRSR (SEQ ID NO:39) (10E),optimized E^(rns) peptide biotin-GRQLRRAGRRLRRRSR (SEQ ID NO:40) (10F),HRSV-G type A peptide biotin-KRIPNKKPGKKTTTPTKKPTIKTTKKDLKPQTTKPK (SEQID NO:41) (10G) and HRSV-G type A biotin-KRIPNKKPGKKT (SEQ ID NO:42)(10H).

FIGS. 11A and 11B. Distribution of FITC-labeled oligonucleotide (32 nt)(11A) and FITC-labeled oligo coupled to optimized E^(rns) peptide (11B)at 56 μg oligo/ml after 30 minutes incubation with cells.

FIG. 12. Passage of HRP through epithelial cell sheet incubated with 0,0.5, 5 and 50 μg/ml BVDV E^(rns) peptide.

DETAILED DESCRIPTION OF THE INVENTION

For diagnostics, the invention provides a peptide comprising an aminoacid sequence derived from E^(rns) of a pestivirus, wherein the aminoacid sequence has, for example, a length of 37 amino acid residuescorresponding to the C-terminus of E^(rns), which is located C-terminalto the RNase domain. Preferably, the amino acid sequence comprises atleast the amino acid residues 191-227 of Pestivirus E^(rns) and has atmost 4 amino acid differences therewith. Preferably, the pestivirusE^(rns) peptide is selected from the group consisting of Classical SwineFever Virus (CSFV), strain Alfort 187, BVDV-1 strain M96751, BVDV-2, orBDV, strain X818. The amino acid sequence preferably comprises a memberselected from the group consisting of:

TABLE 1 CSFV ENARQGAARVTSWLGRQLRIAGKRLEGRSKTW (SEQ ID NO:12) BVDV-1EGARQGTAKLTTWLGKQLGILGKKLENKSKTW (SEQ ID NO:13) BVDV-2EGARVGTAKLTTWLGKQLGILGKKLENKTKAW (SEQ ID NO:14) BDVENARQGAAKLTSWLGKQLGIMGKKLEHKSKTW (SEQ ID NO:15).

More preferably, a member selected from the group consisting of:

TABLE 2 CSFV ENARQGAARVTSWLGRQLRIAGKRLEGRSKTWFGAYA (SEQ ID NO:16) BVDV-1EGARQGTAKLTTWLGKQLGILGKKLENKSKTWFGAYA (SEQ ID NO:17) BVDV-2EGARVGTAKLTTWLGKQLGILGKKLENKTKAWFGAHA (SEQ ID NO:18) BDVENARQGAAKLTSWLGKQLGIMGKKLEHKSKTWFGANA (SEQ ID NO:19).

Such as a member selected from the group consisting of:

TABLE 3 CSFV DTALYLVDGMTNTIENARQGAARVTSWLG (SEQ ID NO:20)RQLRIAGKRLEGRSKTWFGAYA BVDV-1 DTTLYLVDGLTNSLEGARQGTAKLTTWLG (SEQ IDNO:21) KQLGILGKKLENKSKTWFGAYA BVDV-2 ETAIQLLDGATNTIEGARVGTAKLTTWL (SEQID NO:22) GKQLGILGKKLENKTKAWFGAHA BDV DTALYVVDGVTNTVENARQGAAKLTSWLG (SEQID NO:23) KQLGIMGKKLEHKSKTWFGANA.

It is preferable that the peptide is capable of adopting the tertiarystructure of its counterpart in the corresponding E^(rns) protein whenit relates to an antigenic substance, or a precursor thereof, whichallows discrimination between or identification of different pestivirustypes or subtypes, or allows discrimination between or identification ofantibodies against different pestivirus types or subtypes, whichantigenic substance or precursor thereof comprises a peptide as definedherein.

A peptide, antigenic substance or precursor thereof as defined hereinmay be used in diagnosis of Pestivirus infections. This invention alsoprovides a diagnostic test kit for the detection of Pestivirus, orantibodies against Pestivirus types or subtypes, which test kitcomprises a peptide, antigenic substance or precursor thereof as definedherein, together with suitable means for detection. The test kitpreferably provides for an enzyme-linked immunosorbent assay.

The invention also provides a method for the detection of (antibodiesagainst) Pestivirus comprising contacting a sample of a body fluid witha peptide, antigenic substance or precursor thereof as defined herein,in a manner such that a complex comprising the peptide, antigenicsubstance or precursor, and an antibody directed against the peptide,substance or precursor can be formed, followed by detection of thecomplex.

Furthermore, the invention provides a pharmaceutical composition orvaccine for the prophylaxis of Pestivirus infections comprising apeptide, antigenic substance or precursor thereof as defined herein,together with a suitable adjuvant or excipient for administration to amammal. The invention also provides a method for the prophylaxis ofPestivirus infections comprising administering to a mammal a compositionas defined above, in an amount sufficient to elicit an immune responseagainst Pestivirus.

Furthermore, the invention provides a peptidomimeticum that mimics apeptide as defined herein.

Another aspect of this invention is a method for inducing antibodiesagainst Pestivirus types or subtypes comprising administering to amammalian host an antigenic substance or precursor thereof as definedherein, together with a suitable adjuvant, and harvesting resultingantibodies or antibody-producing cells from the mammalian host.

An antibody directed against a type or subtype of Pestivirus obtainableby the above method is also part of the invention. Preferably, theantibody is a monoclonal antibody.

In another aspect, the invention provides a diagnostic test kit for thedetection of or the discrimination between (antibodies against) subtypesor types of Pestivirus comprising the above antibody and suitable meansfor detection.

For antibacterial and transport activity, the invention provides asimilar amino acid sequence as listed in Tables 1 and 2. Systematicanalysis showed that shorter peptides comprising E^(rns) amino acids194-220 had higher transport activity and lower hemolytic activity. Thisamino acid sequence preferably comprises a member selected from thegroup consisting of:

TABLE 4 CSFV RQGAARVTSWLGRQLRIAGKRLEGRSK (SEQ ID NO:1) BVDV-1RQGTAKLTTWLGKQLGILGKKLENKSK (SEQ ID NO:2) BVDV-2RVGTAKLTTWLGKQLGILGKKLENKTK (SEQ ID NO:3) BDVRQGAAKLTSWLGKQLGIMGKKLEHKSK (SEQ ID NO:4)or those presented in Table 5 relating to the L3 loop ofribosome-inactivating proteins.

Most peptides that have been used in serology represent continuousepitopes. It is impossible to detect antibodies against complexdiscontinuous epitopes using small linear peptides and it is difficultto predict discontinuous epitopes based on the amino acid sequence of aprotein. In addition, the antigenic surface of large globular proteinscannot be mimicked accurately with a small linear peptide. We solvedthis problem by predicting an independently folding region in theE^(rns) protein of pestiviruses that adopts a stable tertiary structurewhile retaining its antigenicity. This prediction is crucial for thecorrect design of a useful antigen. Two stretches of Pestivirus E^(rns)show sequence homology with ribonuclease Rh (RNase Rh), a new class ofmicrobial ribonuclease of Rhizopus niveus, a member of the T₂/S RNasesuperfamily. A typical feature for this type of RNase is the low basespecificity and the large molecular weight. The crystal structure ofRNase Rh has been determined (Kurihara et al., J. Mol. Biol. 255:310-320, 1996) and the three-dimensional (3D) structure confirmed thatboth stretches with sequence homology to E^(rns) constitute the activesite of the RNase. Apart from the two stretches of sequence homology,further homology in the rest of the protein was not apparent.

Despite a low sequence homology, we were able to construct an alignmentusing different types of scoring matrices and multiple sequencealignment of a large set of RNase sequences. A satisfactory alignmentwas not possible using alignment software with any parameter setting.Therefore, a part of the alignment was edited manually. For parts withlow sequence homology, the alignment was guided by secondary structureprediction of the PHD software (Rost, B. and Sander, C., 1992, Nature,360:540).

After inspection of the multiple sequence alignment, some majordissimilarities between the sequences of pestivirus E^(rns) and theother RNases can be observed. Compared with sequences of the otherRNases, the pestivirus sequences have a truncation at the N-terminus,large insertions after residues 83 and 135 and an elongated and verydissimilar C-terminus. The 37 C-terminal residues could not be alignedwith the other RNases. Other characteristics of the C-terminus are thehigh number of positive charges and a high score for amphipathichelicity. A helical wheel representation of residues 191-221 shows anamphipathic helix with a hydrophobic face and a positively charged face(FIG. 2). The only three residues that may not correspond with theperfect amphipaticity are Ile210, Arg214 and Arg218. Although no obviousdomains were found with software like SMART (Schultz et al., PNAS 95:5857-5864, 1998), this C-terminal region, which is separate from theRNase domain according to the alignment, with its typical secondarystructure can now be considered as a separate domain or module. Such apositively charged domain in an RNase molecule is not unique for E^(rns)but has also been observed in type II ribotoxins, another class ofRNases. This class of RNases comprises extracellular cytotoxins thathydrolyze the large ribosomal RNA (22) and are able to translocateacross phospholipid bilayers (23). Although ribotoxins are known toenter cells, it is not known which region of the protein is responsiblefor translocation. The type II ribotoxins like alpha-sarcin andrestrictocin contain a large inserted L3 loop (residues 53-91) comparedwith other RNases of the T1 superfamily (24, 25). This loop hasstructural similarity (but no sequence similarity) to loops found inlectin sugar-binding domains and may be responsible for the ribotoxin'sability to bind to the cell surface (24). The C-terminal domain ofE^(rns) has approximately the same length and contains similar sequencemotifs as the ribotoxin II L3 loop (FIG. 3). Although the sequencesimilarity between the ribotoxin L3 loop and the C-terminus of E^(rns)is low (FIG. 3), it is higher than the sequence similarity between L3and the structurally similar lectin-binding domains (24). Although theribotoxin L3 loop is also positively charged, it has no apparentamphipathic character. Another interesting homology of the E'sC-terminal region is with the membrane interacting peptide magainin. Thecenter of the E's peptide has high sequence homology with the N-terminalhalf of magainin (FIG. 3). This homology is even higher compared to thehomology of magainin with other pore-forming peptides that have beendescribed (26)

The (overall) 3D structure of E^(rns) is similar to RNase Rh except forthe C-terminal region, which is surprisingly similar to loop L3 ofrestrictocin or other ribotoxin II proteins. (This protein module likelyfolds independently, is metastable and can change to an alpha helicalstructure when it binds to the cell membrane.) The 3D structure of theC-terminal domain is not very important because of its spatialindependence from the RNase domain.

With the aid of a modular structure, it is possible to define antigenicregions on the surface of the protein which can be mimicked by singlelinear peptides. The domain corresponding to the C-terminal 37 residues(191-227) is the best candidate because of its location on the outer rimon the surface of the E^(rns) dimer; it forms a small functional domainwhich folds independently from the rest of the protein and it is notmasked by any potential carbohydrate.

Development ELISA

The invention provides an antigenic and, in essence, proteinaceoussubstance for discrimination of infected animals with different types ofpestiviruses from animals vaccinated with a subunit vaccine that doesnot contain the E^(rns) peptide and infected animals. The antigenicsubstance is a peptide that corresponds to a C-terminal amino acidsequence of Pestivirus E^(rns) which does not align with RNase Rh butwith an L3 loop of a ribotoxin and folds independently from the rest ofthe protein.

An antigenic substance according to this invention is to be interpretedas any peptide-like or peptide-based substance (e.g., a protein moduleas provided herein optionally linked to another group such as a peptideor protein) capable of inducing an immune response against pestivirus orbeing recognized by serum-containing antibodies against a pestivirus.Precursors of such antigenic substances are, for example, comparablepeptide-like or peptide-based substances which are not immunogenicthemselves but need, for instance, to be coupled to a carrier to be ableto induce an immune response or to be recognized. Peptide-based orpeptide-like substances are intended to include anything with thefunction of the peptides according to the present invention. This meansthat these substances may be peptides themselves in which a number ofamino acid residues have been replaced or modified. It also means thatthey may be fusion proteins, for instance, designed to present the aminoacid sequence of the peptides of the invention on their surface. Thedefinition also includes peptidomimetics and anti-idiotype antibodiesderived from the peptides according to the invention.

In a preferred embodiment, the invention provides peptides that can beused in diagnostic assays for detection of antibodies directed againstspecific pestivirus types (CSFV, BDV, BVDV-I, BVDV-II).

The provision of the protein module, the independently folding region ofE^(rns), relates to all types of pestiviruses and beyond. As aconsequence, the invention is not limited to the peptides specificallydisclosed herein, but extends to analogous peptides and theirderivatives in all types of pestiviruses and all subtypes of theseviruses and to homologues of the L3 loop of (ribosome inactivating)ribotoxin II proteins. Preferred peptides to be used according to theinvention comprise at least antigenic parts of the peptides given inTable 2 or derivatives thereof, their length being from about 27residues up to about 51 residues or transport peptides according to anyone of Tables 1-5.

We have evaluated the applicability of the peptides in diagnostics bythe development of different diagnostic assays: indirect ELISAs in whichthe antigen is recognized in solid phase and an indirect ELISA in whichthe antigen is recognized in liquid phase. Other diagnostic assays can,of course, be easily designed by the man skilled in the art. These may,of course, be provided in any suitable format.

Assays can be performed in solution and on solid phases. They can beperformed using any kind of label, such as enzymes, solid particles,such as metal sols, or other sols, latex particles, dyes, fluorescentsubstances or radioactive materials. They even may be performed withoutlabels, as can be done by agglutination assays. The peptides can be usedto detect antibodies in, for instance, a fluid from a mammal, such asblood, serum, urine, and milk. Usually, the antibody is bound by apeptide, module or substance according to the invention, which may bepresent on a solid phase or in a liquid phase. Afterwards, the complexof peptide and antibody may be detected by a labeled reagent, which canbe a labeled antibody directed against the host's (such as swine, bovineor sheep) antibodies.

According to the invention, the peptides can also be used to obtainantibodies which are specific for Pestivirus types and/or subtypes. Thepeptides are administered to a mammal, usually a rodent, in animmunogenic form, and after one or more booster administrations, theserum from the animal is harvested and antibodies can be purifiedtherefrom.

Alternatively, the spleen of such animals may be removed to obtainantibody-producing cells. These can be changed, by fusion ortransformation, into cell lines producing monoclonal antibodies. Assaysbased on (monoclonal) antibody-directed Pestivirus and induced by thepeptides according to the invention are, therefore, also a part of theinvention.

The peptides according to the invention can, of course, also be used invaccines to prevent infection with Pestivirus. They may be used inconjunction with other antigens to elicit an immune response againstPestivirus. Usually, the peptide has to be coupled to a carrier to bepresented in an immunogenic form before administration to a host. Otherways of rendering a peptide sufficiently immunogenic are known to theperson skilled in the art. Adjuvants are usually added to vaccines toboost the immune response in a more aspecific manner.

Antibacterial and Transport Peptide

The invention further provides a membrane-active peptide or module orsubstance which can be used as an antibiotic and can be used as atransport peptide which is, for example, capable to carry cargos acrossthe cell membrane. For coupling of cargo such as macromolecules, such asdrugs, use is preferably made of a free hydroxyl group when available onthe compound to be bound.

To use transport peptides as a drug-delivery system, these peptides canbe linked to drugs via linkers. From the side of the drug or compound,for instance, cyclosporine A, acyclovir, or terbenafine, a functionalgroup like the hydroxyl group can be used to couple a linking group viaan ester bond. In the linking group, a function like a secondary amine(—NH—) can be inserted, which, by its orientation, catalyzes thecleavage of the ester bond with the drug and subsequently releases theoriginal drug. An example of such a linker is: (drug)—O—CO—CH₂—NR—CO—CH₂—NH—CH₂—CO-link2-(transport peptide).

The transport peptide can be coupled to this linker by a second linker(link2) using different chemistries. For instance, ethylenediamine canbe coupled to the free carboxylic acid of the first linker.Subsequently, the resulting amino group can be coupled to a bromoaceticacid and this bromoacetyl group can react in high yield with, forinstance, the free —SH group of a cysteine in the transporter peptide.Alternatively, the transport peptide can also be coupled directly to thelabile linker via, for instance, a free amino group from the transportpeptide.

Substituents in the linker on the different groups, like the group R onthe tertiary amine, can help to regulate further the stability of theester. Alternatively, thioesters can also be used as an even more labilelinking group.

Alternative strategies can be used for the coupling of the transporterpeptide to compounds that have no easily accessible hydroxyl groupavailable. In the case of terbenafine, an ethynyletheen function can,for example, be used for the addition of the free —SH group in thelinker to form for instance: (drug)—S—(CH₂) n—NR—CO—CH₂—NH—CH₂—CO-link2-(transport peptide). This conjugate caneasily be cleaved under basic conditions (internal base is present assecondary NH-group), releasing the original drug, here, in the example,terbenafine. The membrane-active peptide is similar to the describedantigenic peptide which corresponds to a C-terminal amino acid sequenceof Pestivirus E^(rns) which does not align with RNase Rh but does alignwith magainin and, to some extent, with the L3 loop of ribotoxins andfolds independently from the rest of the protein. The L3 loop peptidesare specifically membrane-active peptides according to the invention aswell. A membrane-active substance according to this invention comprises,for example, a peptide-like or peptide-based substance capable ofinducing leakage of a bacterial membrane or disturbance of a eukaryoticcell membrane without leakage. Peptide-based or peptide-like substancesare intended to include anything with the function of the peptidesaccording to the present invention. This means that these substances maybe peptides themselves in which a number of amino acid residues havebeen replaced or modified. It also means that they may be fusionproteins, for instance, designed to modify the cell specificity of thepeptide. Preferred peptides to be used according to the inventioncomprise at least the membrane-active part of the peptides given inTables 4, 5 and 10. Derivatives with a higher translocation activity arelisted in Table 9.

The invention relates to a set of pestivirus diagnostic assays,antibacterial peptide and transport peptide based on peptidescorresponding to the C-terminal domain of pestivirus E^(rns) or the L3loop of ribotoxin type II proteins. Preferred regions used forpeptide-based diagnostics are listed in Table 2 and preferredmembrane-active peptides used for antibiotics or transport peptides arelisted in Tables 4 or 5, and 9 to 11. However, they can, of course, beused interchangeably for their various uses. The length of the peptideto be used in a diagnostic assay or vaccine is conveniently the exactlength of the domain (residues 191 to 227, 37 residues) but can, ofcourse, be shorter or longer, as long as it does not essentially changein antigenic or immunogenic character. The maximum length of a suitablepeptide (residues 177 to 227, 51 residues) can incorporate a 14-residuelinker region between the RNase domain and the C-terminal domain. Thislinker region may be exposed in case of uncertainty of the exact spatialposition of the C-terminal domain relative to the RNase domain andbecause of the conformational change of the C-terminal domain. For thatreason, the linker region may be part of a large C-terminal antigenicsite. The preferred minimum length of a suitable peptide to be used in adiagnostic assay or vaccine is the part of the C-terminal domain thatforms an amphipathic helix. This is the part of the C-terminal domainwithout the 5 C-terminal hydrophobic residues (191 to 222, 32 residues).

The diagnostic assays based on the peptides can be used to determineantibody levels in blood, serum, milk or other body fluids.

The materials according to the invention can be used for incorporationin vaccines, for example, to provide for a carrier of the desiredantigen-to-antigen presenting cells, or to present an antigen within thecontext of MHC (I or II) peptide presentation, or to provide for mucosalvaccination by providing translocation of a desired antigen over anepithelial (gut) layer. The peptide can also be used to transportvarious cargos in eukaryotic cells, as far as into the Golgi system orin the nucleus of cells. Such cargos can comprise protein or peptidematerial, PNA, RNA or DNA, drugs, secondary metabolites, and so on.Peptide mixtures could be used as well to provide for synergy inantibacterial activity, transportation or translocation.

EXAMPLES Structure Analysis of Pestivirus E^(rns)

A detailed analysis of the primary structure and homology modeling ofpestivirus E^(rns) allowed the definition of antigenic regions on thesurface of the protein which can be mimicked by single linear peptides.The C-terminal 37 residues (191-227) are the best candidates because oftheir location on the outer rim on the surface of E^(rns) and becausethey fold independently from the rest of the protein as a subdomain andare not masked by any potential carbohydrate. The independent characterof the C-terminus is also illustrated by the functional analysis ofE^(rns). E^(rns) mutants have been made which are truncated from residue168. In these mutants, the whole C-terminal part from residues 169 to227 is missing. This mutant is still able to fold natively because thediscontinuous active site is still intact and the mutant has wild-typeRNase activity. Furthermore, the C-terminal 37 residues don't align withthe other RNases but they do align with an L3 loop in ribotoxin type IIproteins and with membrane-active peptides like magainins. Thesemembrane-active peptides have a well-defined function, and for themagainins, it has been shown that they adopt helical conformations ifthey contact the cell membrane. We have demonstrated the membrane-activeproperty of the E^(rns) and/or L3. The membrane-active properties of theE^(rns) peptide agree with the functionally independent nature of thesubdomain. The location of this subdomain, the possibly independentfolding of the sequence, the lack of potential glycosylation sites andits biological function make a peptide representing this region asuitable candidate to be used as an antigen/immunogen for immunoassaysand vaccines. Furthermore, the biological activity of the E^(rns) or theL3 peptide makes these peptides suitable candidates to be used as anantibacterial agent and/or a transport peptide.

ELISA Development

Peptide Synthesis

Peptides were selected from the C-terminal region (residues 191-227) ofCSFV E^(rns), strain Alfort 187, BVDV E^(rns), strain M96751 and BDVE^(rns), strain X818 and the L3 loop of restrictocin (residues 59-88;Lamy and Davies, NAR 19: 1001-1006, 1991) and magainin (Zasloff, PNAS84: 5449-5453, 1987):

CSFV: acetyl-ENARQGAARV TSWLGRQLRI AGKRLEGRSK TWFGAYA-COOH (SEQ IDNO:16) CSFV: biotin-ENARQGAARV TSWLGRQLRI AGKRLEGRSK TWFGAYA-COOH (SEQID NO:16) BVDV: acetyl-EGARQGTAKL TTWLGKQLGI LGKKLENKSK TWFGAYA-COOH(SEQ ID NO:17) BVDV: biotin-EGARQGTAKL TTWLGKQLGI LGKKLENKSKTWFGAYA-COOH (SEQ ID NO:17) BDV: biotin-ENARQGAAKL TSWLGKQLGI MGKKLEHKSKTWFGANA-COOH (SEQ ID NO:19) restrictocin:biotin-GNGKLIKGRTPIKFGKADCDRPPKHSQNGMGK-NH₂ (SEQ ID NO:5) magainin:biotin-GIGKFLHSAGKFGKAFVGEIMKS-NH₂ (SEQ ID NO:24)

Peptides were synthesized according to standard procedures on an AppliedBiosystems 430A synthesizer using Fastmoc chemistry (Fields et al.,Pept. Res. 4: 95-101, 1991). An extra CSFV and BVDV peptide weresynthesized which were N-terminally biotinylated instead of acetylated.

Serum Samples

The following swine serum samples were incorporated in the study toevaluate the peptide ELISAs.

-   -   Negative field serum samples (n=96) were randomly obtained from        slaughtered adult pigs. Sera were all tested negative in the        CSFV-E2 and pan-pestivirus antibody-specific Ceditest ELISAs.    -   Pestivirus serum antibody-positive but CSFV-negative serum        samples (n=96) were randomly obtained from slaughtered adult        pigs. Swine sera were tested negative in Ceditest        CSFV-E2-specific ELISA (Colijn et al., Vet. Micro. Biol. 59:        15-25, 1997) and positive in the pan-pesti ELISA (Paton et        al., J. Virol. Meth. 31: 315-324, 1991; Kramps et al., Vet.        Micro. Biol. 64: 135-144, 1999).    -   CSFV antibody-positive field serum samples tested by virus        neutralization test were obtained (n=95) from an infected pig        farm (VR) that was infected during the CSF epizootic in the        Netherlands in 1997-1998.    -   Sequential serum samples were collected during a        vaccination/challenge experiment of 12 pigs that were vaccinated        with E2 and infected with CSFV, strain Brescia. Specific        Pathogen-Free (SPF) animals were challenged with the virulent        CSFV strain Brescia two weeks after a single vaccination with        the E2 subunit vaccine.    -   Panel of swine sera that were experimentally infected with BVDV        (n=5, numbers 4-8).    -   Panel of swine sera that were experimentally infected with CSFV        strain Paderbom (n=5, numbers 9-13).    -   Panel of bovine sera that were experimentally infected with BVDV        (n=9, numbers 1-6, r4590-51, r4590-52, 841).    -   Reference panel obtained from the European reference laboratory        for CSFV: sera from swine that were experimentally infected with        CSFV (n=14), BVD (n=1) or BVDV (n=12). Three sera were obtained        from swine with experimental mixed infections of BVDV/BDV (n=1)        and CSFV/BVDV (n=2).    -   Pool of hyperimmune sera against CSFV (HIS CSFV).    -   Pool of hyperimmune sera against BVDV (HIS BVDV).

Solid Phase Peptide ELISA (sp-ELISA)

Test Procedure

For the sp-ELISA, a similar format was chosen as for a previouslydeveloped RSV G-peptide ELISA (Langedijk et al., J. Imm. Meth. 193:153-166, 1996). One microgram of N-terminally acetylated pestiviruspeptide was coated per well of a high binding capacity flat bottommicroplate (Greiner) in 50 μl of carbonate buffer, pH 9.0, at 37° C. anddried overnight. The optimal dilution of the peptide to coat ELISAplates was chosen in such a manner that maximum binding was obtained asdetermined in a checkerboard titration. Test sera were titrated.Mouse-anti-swine IgG (23.3.1b) conjugated to horseradish peroxidase(HRP) was diluted 1:1000. Rabbit anti-bovine IgG-HRP (P0159, Dako,Denmark) was diluted 1:1000. Conjugates and test sera were incubated forone hour at 37° C. in ELISA buffer (8.1 mM Na₂HPO₄, 2.79 mM KH₂PO₄, 0.5M NaCl, 2.68 mM KCl, 1 mM Na₂EDTA, 0.05% v/v Tween 80, pH 7.2)containing 4% horse serum. The substrate chromogen consisted ofABTS/H₂O₂. Incubation was performed during 30 minutes at 22° C. OD wasmeasured at 405 nm (Titertek multiscan).

Results

The reactivity of BVDV-positive swine sera (4-8) and CSFV-positive swinesera (9-13) were tested for reactivity in the CSFV sp-ELISA and the BVDVsp-ELISA.

The reactivity of bovine sera (numbers 1-6, r4590-51, r4590-52, 841)were tested for reactivity in the CSFV sp-ELISA and the BVDV sp-ELISA.

Reactivity of the sera with the peptides was excellent, which shows thatthe peptides indeed correspond to an immunodominant region of E^(rns).This agrees with the prediction of the immunodominant character of thesubdomain. However, the CSFV and BVDV sera are cross-reactive for bothpeptides. Although the panel of CSFV-specific swine sera reacted betterthan the panel of BVDV-specific swine sera in the CSFV ELISA, thereactivities of both panels of sera are similar in the BVDV ELISA.Similarly, the panel of BVDV-specific bovine sera shows high reactivityin the BVDV peptide ELISA (FIG. 5D), but the sera also cross-reactconsiderably in the CSFV ELISA (FIG. 5C).

Liquid Phase Peptide ELISA (1p-ELISA).

Because of the high cross-reactivity in the solid phase peptide ELISA,an ELISA was developed in which the antigen was recognized in liquidphase (1p-ELISA). Moreover, by labeling the homologous peptide of thepestivirus of interest (CSFV peptide), unlabeled heterologous peptide ofthe cross-reactive pestivirus (BVDV peptide) could be used to blockunspecific cross-reactivity.

In the liquid phase peptide ELISA for detection of antibodies againstCSFV, the test serum was incubated with a mixture of biotinylated CSFVpeptide and acetylated BVDV peptide (without biotin). CSFV-specificantibodies will preferably bind the biotinylated CSFV peptide andBVDV-specific antibodies will preferably bind the nonbiotinylated BVDVpeptide. Subsequently, the mixture is transferred to an avidin-coatedmicrotiter plate and the antibodies complexed to the biotinylated CSFVpeptide will be caught by avidin and can be detected with an anti-swineperoxidase conjugate and subsequent incubation with substrate.

Test Procedure:

Avidin-coated microtiter plates: 400 ng of ImmunoPure avidine (No.21121, Pierce, Rockfort, Ill., USA) in 100 μl of carbonate buffer (pH 9)in each well of a high binding capacity flat bottom microplate(Greiner). Plates were covered and incubated overnight at 37° C. Aftercoating, the plates were kept frozen until use.

Before use, the avidin-coated plates were incubated with 100 μl ofphosphate-buffered saline (PBS, pH 7) with 10% horse serum per well fortwo hours at 37° C. on a shaker.

Meanwhile, test serum (1:50) was incubated with a mixture of 10 ngbiotinylated CSFV peptide and 30 ng of BVDV peptide in 100 μl of ELISAbuffer with 4% of horse serum for one hour at 37° C.

Avidin-coated plates were washed and 100 μl of test serum and peptidemixture was transferred in the wells and incubated for 45 minutes at 37°C. Subsequently, plates were washed and incubated with 100 μlmouse-anti-swine IgG (23.3.1b) conjugated to horseradish peroxidase(HRP) (Van Zaane et al., 1987) diluted 1:1000 or with rabbit anti-bovineIgG-HRP (P0159, Dako, Denmark) diluted 1:1000. The substrate chromogenconsisted of ABTS/H₂O₂. Incubation was performed during 30 minutes at22° C. OD was measured at 405 nm (Titertek, multiscan). Cutoff value waschosen at OD>0.5, which is approximately 3 times the average backgroundof known negative sera.

Results

The reactivity of BVDV-positive swine sera (4-8) and CSFV-positive swinesera (9-13) were tested for reactivity in the CSFV 1p-ELISA (FIGS.5A-5D). This test format showed much better specificity than thesp-peptide ELISA.

To determine the specificity of the 1p-peptide-ELISA, 96 negative fieldserum samples were tested in the 1p-peptide-ELISA for CSFV E^(rns)-Ab.Only 2 of 96 samples showed a positive response (cutoff was chosen atOD>0.5). Based on these data, the specificity of the 1p-peptide ELISAfor CSFV E^(rns)-Ab amounts to 98% (=94/96×100%) (FIGS. 6A-6B).

To determine the specificity of the 1p-peptide-ELISA, 96 field sera thatcontain antibodies directed against other pestiviruses than CSFV (BVDVand BDV) were tested in the 1p-peptide-ELISA. Only 2 of 96 samplesshowed a positive response (OD>0.5). Based on these data, thespecificity of the 1p-peptide ELISA for CSFV E^(rns)-Ab fornon-CSFV-pestivirus-positive sera amounts to 98%(=94/96×100%) (FIGS.6A-6B).

To determine the sensitivity of the 1p-peptide-ELISA, 95 field serumsamples from a CSFV-infected farm (VR) obtained during the CSF epizooticin the Netherlands in 1997-1998 were tested in the 1p-peptide-ELISA. Nota single serum sample showed a positive response (OD>0.5). Based onthese data, the sensitivity of the 1p-peptide ELISA for CSFV antibodiesamounts to 100% (FIGS. 6A-6B).

An interesting application of the 1p-peptide ELISA is a diagnostic testthat can be used to detect CSFV infection of E2-vaccinated pigs.Therefore, sera of E2-vaccinated pigs should be unreactive in the1p-peptide ELISA and should be positive after CSFV challenge of thepigs. Successive serum samples, which were collected during a challengeexperiment of 12 E2-vaccinated pigs that were infected with CSFV, weretested in the 1p-peptide ELISA (FIG. 7). The results show that all serabut one from E2-vaccinated pigs were negative prior to CSFV challenge.All animals (except one) seroconverted 14 to 28 days after challenge.Finally, the performance of the 1p-peptide ELISA was compared with theE2-based Ceditest ELISA and two other E^(rns)-based ELISAs. Thereactivity of a panel of European reference sera (see methods) wastested in all four ELISAs (Table 6). Although the E2-based ELISA wassuperior (one false-negative), the 1p-peptide ELISA performed better(three false-negative) than the other ELISAs based on epitope blockingusing complete E^(rns) (5 false-negative, one false-positive, onefalse-negative and six false-positive). It is very likely that the1p-peptide ELISA can even be optimized when it is changed into anantibody-blocking format, like the other three ELISAs in Table 6.

To illustrate the compatibility of the peptide ELISA with otherpestiviruses, the CSFV-specific peptide ELISA was changed into a BVDVand a BDV ELISA by exchanging the biotinylated CSFV peptide for abiotinylated BVDV peptide or a biotinylated BDV peptide and theacetylated BVDV peptide for the acetylated CSFV. The same amount ofpeptide was used as in the 1p-CSFV peptide ELISA and all assayconditions were kept similar. The panel of swine sera that wereexperimentally infected with BVDV (n=5, numbers 4-8) or CSFV (n=5,numbers 9-13) were tested in the three different 1p-peptide ELISAs forthe three different pestivirus types. Table 7 shows that BVDV-positivesera react best in the BVDV-specific peptide ELISA and that theCSFV-positive sera react best in the CSFV ELISA, although the CSFV seracross-react to some extent with the BVDV peptide. As expected, on thebasis of the sequence homology, the BVDV and BDV ELISAs show lessdifferentiation. The BVDV and BDV ELISAs both contained acetylated CSFVpeptide as competing antigen. Some improvement may be possible when alsoacetylated BDV and BVDV peptide would be used as competing antigen inthe BVDV and BDV ELISAs, respectively.

The acetylated CSFV peptide (Table 2) was further used to test theimmunogenicity of the peptide in pigs and to examine whether vaccinationwith the peptide could protect the pigs after CSFV challenge. Pigs werevaccinated with the various amounts of the peptide, formulated inFreund's Complete Adjuvant (FCA). Four weeks later, the pigs werevaccinated again with 1.3 mg peptide, formulated in Freund's IncompleteAdjuvant (FIA). Five control pigs were vaccinated similarly with justFCA and FIA. Three weeks after the second vaccination, the pigs werechallenged nasally with 100 LD50 CSFV, strain Brescia 456610. Antibodyreactivity against the peptide was monitored during the experiment.Before and after challenge, virus isolations were performed on whiteblood cells. After death or euthanasia, organs (tonsil, spleen, kidneyand ileum) were tested for the presence of viral antigen using animmunofluorescence test.

Monoclonal Antibody Production

Production of E^(rns) peptide-specific monoclonal antibodies (Mabs) wasperformed as described (Wensvoort et al., 1986). Two BALB/c mice wereimmunized intraperitoneally with 400 μg CSFV or BVDV peptide (residues191-227), mixed with Freund's complete adjuvant (FCA). After four weeks,the mice were boostered with 400 μg of peptide mixed in incomplete FCA,and three weeks later, the mice were boostered with 400 μg of peptide inphosphate-buffered saline. Three days later, the spleen cells were fusedwith sp20 cells and hybridomas were grown in selective medium. TheE^(rns) specificity of the produced Mabs (Mab 906-2-1 (BVDV) and Mab907-35-1-1 (CSFV)) was determined using an E^(rns) antigen detectionELISA. Additionally, the Mabs reacted in the E^(rns) peptide-basedELISAs.

Transport Activity of E^(rns) Peptide

Test Procedure.

Binding of E^(rns) peptide. Monolayers or cytospins of cell suspensionsof several cell types (Ebtr, SK6, sf-21, Caco-2 and HT-29) were fixedwith acetone or 4%-paraformaldehyde. Glass slides or coverslips withfixed cells were incubated with biotinylated CSFV peptide (100 or 10μg/ml PBS) for one hour at 37° C. After washing with PBS, the slips wereincubated with avidin-HIRP (1:100, Zymed) or avidin-FITC (1:70, Zymed)for 30 minutes at 37° C. Cells were inspected for specific binding ofthe peptide by light microscopy (HRP) or fluorescence microscopy (FITC).

Translocation of E^(rns) peptide. Translocation of the peptide acrossthe plasma membrane was studied by incubation of live cells insuspension or subconfluent monolayers on coverslips with biotinylatedpeptide (200 to 0.4 μg/ml culture medium) for 1, 10, 30, 45 or 180minutes. After the time period, cells were fixed with 4%paraformaldehyde or cold methanol and labeled with avidin-FITC asdescribed above. Fixed cells were inspected with fluorescencemicroscopy. Internalization was established with confocal microscopy.The following cell lines were used: A72, canine fibroblast tumor cells;MDCK, canine kidney epithelial cells; CCO, sheat-fish ovaria cells;EK-1, eel kidney cells; CHS-E, salmon embryonal cells; BUEC, bovineumbilical endothelial cells; BFDL, bovine fetal diploid lung cells(fibroblast); PUEC, porcine umbilical endothelial cells; HT 29,colorectal adenocarcinoma, colon epithelial cells; CaCo-2, colorectaladenocarcinoma, colon epithelial cells; Hela, adenocarcinoma, cervix;Vero, normal monkey kidney epithelial cells; SK6, swine kidney cells;NPTh, newborn pig thyroid cells; ECTC, embryonal calf thyroid cells;MDBK, normal bovine kidney epithelial cells; EBTr, epithelial bovinetrachea cells; Bovine sperm cells; Sp20 mouse myeloma B-cells.

Transepithelial transport. The potential of E^(rns) peptide topermeabilize epithelium and assist transport of molecules acrossepithelium was tested in USSING chambers of the snapwell type withCaCo-2 or HT29 cells that closely mimic epithelial cell sheets.

The potential of E^(rns) peptide to carry nonlinked molecules acrossepithelium was tested by mixing HRP (0.08 μg/ml culture medium) withseveral concentrations of E^(rns) peptide (50, 5 and 0.5 μg/ml ringersmedium) in the upper chamber. Samples were drawn from the lower chamberafter 15, 30, 45, 60, 120 and 240 minutes, which were tested for HRPconcentration.

Transdermal transport. The potential of the an E^(rns) peptide topenetrate the skin was tested by applying 150 μl 0.3-4 mM ofbiotinylated peptide in a chamber containing an isolated piece of“fresh” human breast skin or in a chamber which was glued on the skin ofa live pig, or applying 50 μl of peptide solution on the skin with acotton wool tip, or applying 50 μl of a peptide solution mixed with 50μl of contact gel on the skin for 30 minutes to 120 minutes. After theincubation time, the pig was killed, the skin was cleaned and biopsied,and a biopsy was frozen in liquid nitrogen. Cryosections of the skinsamples were fixed on microscopic slides with acetone and incubated withstreptavidin-FITC ( 1/100) for 30 minutes.

Hemolytic assay. Hemolytic activity of various peptide concentrationswas determined by incubation with human, guinea pig or sheep erythrocytesuspensions (final erythrocyte concentration, 1% v/v) for one hour at37° C. After cooling and centrifugation, the optical density of thesupernatants were measured at 540 nm. Peptide concentrations causing 50%hemolysis (EC₅₀) were derived from the dose-response curves.

Clonogenicity of mammalian cells. HeLa or EBTr cells were cultured inDMEM, supplemented with 20% fetal bovine serum and antibiotics in ahumidified atmosphere supplied with 5% CO₂ at 37° C. Exponentiallygrowing cells were treated with trypsin and transferred to wells of a96-well microtiter plate, resulting in approximately 300 cells for each30 μl of growth medium containing various concentrations of peptide.After incubation for 75 minutes (the plates were incubated upside downto avoid anchorage), the cells were transferred and plated in wells oftissue culture plates, which contained 100 μl of growth medium. Cellgrowth was checked after 3 to 6 days.

Antimicrobial assay. Two bacterial strains (Escherichia coli ATCC 25922and Enterococcus faecalis ATCC 29212) were inoculated on heart infusionagar with 5% sheep blood and incubated aerobically overnight at 37° C.From the pure cultures, suspensions were made in saline to a density of0.5 McFarland. These suspensions were diluted ten-fold in Mueller HintonII broth, resulting in a final inoculum of approximately 10⁷ cfu/ml.

Standard 96-well microtiter trays were filled with 100 ill of two-folddilutions of peptide in physiologic salt solution in each well,resulting in the following concentration range: 4000, 2000, 1000, 500,250, 125, 62.25, 31.63, 15.82, 7.96 and 0 μg/ml. The trays in column 12were filled with 200 μl MH II broth (negative control).

Columns 1-11 of the microtiter trays were filled with 100 μl of thefinal inoculum of the bacterial suspensions, thus diluting theconcentrations of the peptide two fold, resulting in the followingpeptide concentration range in the wells: 2000, 1000, 500, 250, 125,62.25, 31.63, 15.82, 7.96, 3.98 and 0 μg/ml. In rows B, C and D, 100 μlof the final inoculum of E. coli and, in rows E, F and G, 100 μl of thefinal inoculum of E. faecalis was pipetted. The final bacterialconcentration was approximately 5×10⁵ cfu/well. All trays were sealedand incubated overnight at 37° C. After incubation, the microtiter trayswere inspected visually for bacterial growth and the absorbance of thecultures at 630 nm (A₆₃₀) was determined with an ELISA-reader. Thepeptide concentrations in the wells with the lowest concentrations thatshowed no visible growth or increase in absorbance compared with thenegative control wells were considered to be the Minimum InhibitoryConcentrations (MICs). The experiment was performed independently intriplicate. The test was repeated again in triplicate after two dayswith the final peptide concentration range in the wells: 5000, 2500,1250, 625, 312.5, 156.25, 78.12, 39.06, 19.58, 9.79, 4.88, 2.44 and 1.22μg/ml.

Peptide synthesis. A panel of truncated E^(rns) peptides was synthesizedto elucidate the minimal membrane-active region. A panel of truncatedrestrictocin L3 peptides was synthesized to elucidate the minimalmembrane-active region. Substitutions in the amino acid sequence of thetransport peptide module can be applied to increase the translocationactivity. An optimized transport peptide can, for example, besynthesized according to retro-inverso peptide chemistry, in which thesequence is reversed and D-amino acids are used instead of L-aminoacids. Synthesis is performed as described above.

Coupling of peptide to oligonucleotide. The optimized E^(rns) peptidecontains a Broom-acetic acid at its N-terminus (Broom-GRQLRIAGKRLEGRSK)(SEQ ID NO:25) coupled to two sulfhydryl groups at the 5′- and the3′-end of an FITC-labeled 32 residue long oligonucleotide(Thiol-GT^(FITC)CCACCGAGGCTAGCTACAACGACCCTTATAT-thiol) (SEQ ID NO:26).

Results

To determine the binding of the E^(rns) peptide, biotinylated CSFVpeptide was incubated with various fixed cells. Binding was determinedafter incubation with avidin-HRP or avidin-FITC. Biotinylated CSFVpeptide was able to bind to all tested cell types. There was a markeddifference between the binding to paraformaldehyde versus acetone-fixedcells. In contrast to the paraformaldehyde-fixed cells, theacetone-fixed cells showed less binding with the peptide. Because muchof the membrane fraction gets washed away after acetone-fixation, thissuggests that the peptide may bind to the membrane.

Next, cell suspensions (mouse myeloma and bovine sperm) and subconfluentmonolayers of various cell types (see test procedure) were incubatedwith biotinylated CSFV peptide and fixed after different time intervals.Inspection with fluorescent microscopy and confocal microscopy showedthat the peptide had penetrated inside all cell types. The peptideentered the cell within one minute, and optimal fluorescence wasestablished after 30 minutes (FIG. 8). Peptide was translocated tospecific regions inside the nucleus, which may be the nucleoli. Peptidewas also distributed around the nucleus in membranous parts in thecytosol. The colocalization in the nucleoli was established by doublestaining with acridinorange (Merck, Darmstadt, Germany) andstreptavidin—Texas red. Yellow fluorescence was observed which indicatescolocalization of peptide and nucleoli stain. Next, the membrane-activeregion of the peptide was precisely defined by testing the translocationactivity of a panel of truncations of the E^(rns) peptide and somepeptides with N-terminal additions and C-terminal deletions (Table 8).Translocation to the nucleus was more effective with the CSFV, strainAlfort, E^(rns) peptide compared to the BVDV or BDV E^(rns) peptide. TheCSFV, strain Alfort, E^(rns) peptide translocated also more effectivelythan the CSFV peptide corresponding to strains in which positions 209,210 and 217 were substituted. Furthermore, deletion of the seven mostC-terminal residues and the three N-terminal residues increased thenuclear translocation activity of the peptide. To reduce the length ofthe transport peptide further, the region responsible for translocation(residues 194-220) was truncated while arginines were introduced tomaintain or enhance the translocation activity. N-terminal deletionswere made of 4 and 5 residues and one extra arginine was introduced atthe same face of the presumed helix. These shorter peptides retainedtranslocation activity (Table 9). Next, 11 N-terminal residues weredeleted and the glutamic acid was substituted by arginine. Thissubstitution enhanced the translocation activity by a factor of 33.Replacing the two lysine residues by arginine residues enhanced thetranslocation activity by an additional factor of three. Introducingmore arginines in the sequence did not improve the translocationactivity of the biotinylated peptide (Table 9). Next, the optimizedpeptide (MDK-20) was synthesized with D-amino acids according to theretro-inverso approach with an additional lysine-MTT (peptide A941:biotin-rsrgrlrrgairlqrgK (SEQ ID NO:27) (MTT)-BrHAc). This peptideretained its translocation activity and showed even a highertranslocation activity compared with the original MDK-20, showing theadvantages of the retro-inverso approach.

Because of the homology between the E^(rns) peptide, magainin and the L3loop peptide, the biotinylated magainin and L3 loop peptide were alsotested for translocation activity. L3 showed translocation activity,magainin did not (FIG. 9). Next, the membrane-active region of thepeptide responsible for translocation was precisely defined by testingthe translocation activity of a panel of truncations of the L3 peptide(Table 10). To elucidate general rules for peptide translocation, wesearched for new transport peptides and tested them for translocationacross the plasma membrane.

HIV-1 tat peptide, which is a transcription factor that binds DNA, theAntP peptide, which is a homeobox protein, and transportan, which is ahybrid peptide of a bee venom, and galparan. The only similarity betweenthe known transport peptides is the content of basic residues. See alsoWender, P. A. et al., 2000, Proc. Natl. Acad. Sci. USA, 97:13003-8, who,however, require high >50% arginine content.

To search for more transport peptides, we tested the translocation ofbasic peptides which were derived from linear DNA-binding motifs;RNA-binding motifs; heparin-binding motifs, basic enzymatic cleavagesites and nuclear localization signals. For nuclear localization signals(NLS), it is already known that they are able to translocate the nuclearmembrane, although it is thought that they need to be recognized by areceptor in order to translocate. We tested whether peptidescorresponding to nuclear localization sites could also be used asgeneral transport peptides that cross the plasma membrane.

Several types of short transport peptides were found:

Monopartite: PKKKRKV (SEQ ID NO:28). Binds the import receptor Impalpha/imp beta complex.

This sequence is identical to the NLS of simian virus 40 large Tantigen.

Bipartite: KRPAAIKKAGQAKKKK (SEQ ID NO:29) (lysine-rich signals).

NLS of HIV-1 Rev RQARRNRRRRWR (SEQ ID NO:30).

HIV-1 rev is a viral RNA export factor and shuttles RNA outside thenucleus. The sequence motif binds RNA.

A synthetic peptide has been developed (RSG-1.2) that binds the Revbinding site RNA with 10 times higher affinity

DRRRRGSRPSGAERRRRR (SEQ ID NO:31)

NLS of human herpesvirus-8 K8 protein TRRSKRRSHRKF (SEQ ID NO:32).

K8 protein is homologous to EB1 protein of Epstein-Barr virus (EBV),which is a transcriptional activator which directly binds specific siteson the DNA.

Another class of basic peptides that may in some cases also be NLSpeptides are RNA/DNA-binding peptides. For instance, the HIV-1 Revpeptide also binds directly to RNA.

Other examples of peptides that bind DNA or RNA:

RNA-binding element of flockhouse virus (FHV)

NRTRRNRRRVR. (SEQ ID NO:33)

RNA-binding element of bacteriophage lambda-N

QTRRRERRAEKQAQW. (SEQ ID NO:34)

Another class of basic peptides are basic enzymatic cleavage sites.

An example of a viral enzymatic cleavage site is in the surface proteinsof alpha viruses. The cleavage site between E3 and E2 is highly basic.After cleavage, it is the C-terminal region of E3 which is located atthe distal end of the viral spike, interacting primarily with E2.

Western equine encephalitis virus E3: KCPSRRPKR (SEQ ID NO:35).

Other examples of basic peptides are linear heparin-binding sites. Thetransport peptide corresponding to the E^(rns) C-terminal domain alsobinds heparin. Examples are peptides found in HRSV-G, type A:KRIPNKKPGKKTTTKPTKKPTIKTTKKDLKPQTTKPK (SEQ ID NO:36) and HRSV-G, type B:KSICKTIPSNKPKKK (SEQ ID NO:37).

These peptides showed translocation activity (Table 11). In the case ofthe heparin-binding sites of HRSV-G, the region responsible fortranslocation was mapped (Table 11). One of the most active HRSV-Gpeptides (Biotin-KRIPNKKPKK) (SEQ ID NO:38) was further optimized bychanging all lysine residues into arginine residues to check whether thepeptide showed higher translocation activity when residues wereintroduced which were more basic. Changing the four lysines to argininesimproved the translocation activity by a factor of three (Table 11).

According to Table 9, the most active biotinylated peptide (residues194-220) was still (faintly) detectable at 125 nM. As a result, theelucidation of the minimal essential part of the translocation peptidealso proves that a C-terminal cargo of at least 6 residues can betransported and an N-terminal cargo of at least 13 residues plus abiotin can be transported. Because the peptide is probably also able totransport the RNase domain of E^(rns) inside the cell, it is alsoexpected that large proteins can be transported by the peptide. Aftermixing equimolar amounts of streptavidin-FITC (60 kD, nonglycosylated,neutral) with the most active E^(rns) peptide (residues 194-220) andwith an optimized E^(rns) peptide (biotin-GRQLRIAGKRLEGRSK) (SEQ IDNO:25), it was possible to transport streptavidin-FITC inside the celland the nucleus (FIG. 10). The peptides were also able to carryavidin-Texas Red (66 kD, glycosylated, positively charged) inside thecells. Also, the optimized E^(rns) peptides and the long and shortHRSV-G type A peptides (MDN-12 and MDP-32 according to Table 11) weretested for their ability to transport streptavidin-FITC into the cell(FIGS. 10F-10H). The translocation activity of the peptide complexed tothe streptavidin cargo was different from the translocation of thebiotinylated peptide alone. The optimized E^(rns) peptide MDM-27, whichhas two additional positive charges compared to the optimized E^(rns)peptide MDK-20, transports streptavidin-FITC much better than MDK-20. Incontrast, in the case of the uncomplexed biotinylated peptide, thetranslocation activity was similar. This effect was even more pronouncedwhen the longer HRSV-G peptide (MDN-12) was compared to the shorterHRSV-G peptide (MDP-32), which has less positive charge but highertranslocation activity as a single, uncomplexed molecule without cargo(FIGS. 10G and 10H).

To check whether the transport peptide was also able to transportproteins that retain biological activity, the peptide was complexed withstreptavidin conjugated to the enzyme β-galactosidase (600 kD). Thecomplex was transported efficiently into the cells and retained itsability to release nonreducing terminal galactose. To check whether thepeptides could transport oligonucleotides, the optimized E^(rns) peptidewas activated by a Broomacetic acid. Transport moduleBroom-GRQLRIAGRRLRGRSR (SEQ ID NO:39) was coupled to an FITC-labeled 32oligonucleotide at the 5′- and the 3′-end. The complex was tested fortranslocation inside the cell and the nucleus. Titration of theuncoupled oligonucleotide and the complex of peptide and oligo showedthat the intracellular accumulation of the oligonucleotide was 75 timeshigher when it was coupled to the transport peptide. To check whetherthe membrane destabilizing activity had a general toxic effect on cells,leakage of the cell was tested for tryphan blue after peptide incubationof 30 minutes. Only at high concentrations of peptide (>35 μM), sometryphan blue could be determined inside the cell, especially in isolatedareas in the nucleus.

Hemolysis of erythrocytes can also be indicative for lytic effect of thepeptides on eukaryotic cell membranes. Hemolysis of erythrocytes fromseveral species was tested with the panel of E^(rns) peptides (Table 8).The different peptides show a broad range of hemolytic activities onguinea pig erythrocytes. The peptide with the highest translocationactivity (residues 194-220) has a low hemolytic activity. No significanthemolysis was observed with sheep and human erythrocytes. The effect ofthe E^(rns) peptide on cell growth of HeLa cells and EBTr cells wasdetermined in a clonogenicity assay as shown in Table 12. These datacorrespond to the other toxicity assays and indicate that thetranslocation activity is much higher than the cytotoxic activity.

Transdermal Transport

Next, it was tested whether the optimized E^(rns) peptide (MDK-20) wasable to penetrate an epithelial layer. Because of the stratum corneumlayer, the skin seems to be the most difficult epithelial barrier totake. To test the penetration by the biotinylated peptide, a sample ofhuman breast skin was contacted with the biotinylated peptide in vitroduring two hours. In fixed cryosections of the skin, the biotinylatedpeptide could be visualized with streptavidin-FITC in the epidermis andthe dermis (FIG. 13). The same results were obtained when the peptidewas contacted with the skin of a pig in vivo. As soon as 30 minutesafter application, penetration of the peptide into the epidermis wasobserved. Because of the high amount of proteolytic enzymes in the skin,transdermal transport was also tested with the stable retro-inversopeptide containing D-amino acids. Much more accumulation in the skin wasobserved for the peptide with D-amino acids (A941) compared with theoriginal peptide containing L-amino acids (MDK-20)

Trans epithelial transport. Next, the peptide was tested for the abilityto assist leakage of proteins through an epithelial cell sheet.Horseradish Peroxidase, which was mixed with the peptide, could betransferred through the cell sheet.

Antibacterial activity. Because of the membrane activity and thehomology with antibacterial peptides, the antibacterial activities ofthe peptides were determined as described above. The peptides indeedshowed antibacterial activity against the gram-negative E. coli (Table13) but not against E. faecalis. The MICs against E. coli correlate withthe translocation activity of the peptide. The restrictocin L3 peptideshowed no antibacterial activity.

TABLE 5 Restrictocin GNGKLIKGRTPIKFGKADCDRPPKHSQNGMGK (SEQ ID NO:5)Mitogillin -------------------------------- (SEQ ID NO:5) Toxin AspfI-------------------------------- (SEQ ID NO:5) Alpha-sarcin-D---P----------S---------KD-N-- (SEQ ID NO:6) Gigantin-E--IL----------S---------KD-N-- (SEQ ID NO:7) Clavin-D--IL-------W-NS---------K--D-- (SEQ ID NO:8)

TABLE 6 Comparison of reactivity of reference sera with different CSFVdiagnostic tests. E2-ELISA E^(RNS) ELISA No. DPI¹ Inoculum Cedi-E2²Cedi-Erns³ Bommelie⁴ Peptide 1 43 CSFV Visbek/Han 95 + + + 0.171 2 16CSFV Visbek/Han 95 + + + 0.477 3 20 CSFV Visbek/Han 95 + + + 1.796 4 20CSFV Visbek/Han 95 + − + 3.165 5 14 CSFV Visbek/Han 95 + + + 0.7 6 21CSFV Alfort 187 + − − 0.186 7 29 CSFV Diepholz1/Han 94 + + + 2.25 8 29CSFV Diepholz1/Han 94 + − + 2.619 9 29 CSFV Diepholz1/Han 94 + − + 1.85710 34 CSFV Visbek/Han 95 + − + 3.87 11 55 CSFV Visbek/Han 95 + + + 1.12212 93 CSFV C-strain + + + 0.543 13 69 CSFV Diepholz1/Han 94 + + + 2.14614 28 CSFV Diepholz1/Han 94 − + + 1.103 15 BVDV NADL − − − 0.103 16 BDV− − − 0.45 17 BVDV 2214 − + + 0.161 18 BVDV NADL − − − 0.118 19 BVDVNADL + BDV − − + 0.136 20 BVDV + CSFV + + + 3.208 Alfort 187 21 BVDVOsloss − − − 0.146 22 BVDV Osloss − − − 0.151 23 BVDV Osloss − − − 0.15124 BVDV Osloss − − − 0.162 25 BVDV Osloss − − − 0.172 26 BVDV Osloss −− + 0.164 27 BVDV Osloss − − + 0.149 28 BVDV Osloss − − + 0.163 29 BVDVOsloss − − + 0.215 30 CSFV Alfort 187 + BVDV + + + 0.567 Osloss ¹Dayspost infection. Serum numbers 21-29 obtained from same animal ²Cedi-E2assay registration no.: BFAV/KSP/D10/98 ³Cedi-Erns assay ⁴BommelieAG/Intervet assay

TABLE 7 Comparison of reactivity (OD 405) in different Pestiviruspeptide ELISAs. Serum Number CSFV BVDV BDV  4 BVDV 0.236 1.346 0.781  5BVDV 0.129 0.724 0.388  6 BVDV 0.106 3.211 0.824  7 BVDV 0.250 4.0004.000  8 BVDV 0.104 4.000 2.325  9 CSFV 0.487 0.367 0.349 10 CSFV 2.0901.612 0.343 11 CSFV 2.555 0.610 0.246 12 CSFV 1.133 0.453 0.412 13 CSFV1.253 0.573 0.436 HIS CSFV 1:500 2.263 0.471 0.624 HIS CSFV 1:1000 1.4190.267 0.356 HIS CSFV 1:2000 0.871 0.166 0.180 Negative serum 0.152 0.1500.153 All sera were diluted 1:50 except for the HIS sera

TABLE 8 Determination of minimal membrane-active sequence of E^(rns.)Residue Cellular Nuclear Hemolysis number Sequence¹ fluorescencefluorescence (mg/ml) 191-227 $ENARQGAARVTSWLGRQLRIAGKRLEGRSKTWFGAYA#(SEQ ID NO:16) ++++ ++ 0.07 —COOH  191-227²$ENARQGAARVTSWLGRQLSTAGKRLE*RSKTWFGAYA# (SEQ ID NO:43) +++ + 0.08 —COOH191-227 $ENARQGAAKLTSWLGKQLGIMGKKLEHHSKTWFGANA (SEQ ID NO:44) ++++ +0.25 (BDV) —COOH  194-230³ $EGARQGTAKLTTWLGKQLGTAGKKLENKSKTWFGAYA# (SEQID NO:45) ++++ + 0.25 184-223 $DGMTNTIENARQGAARVTSWLGRQLRIAGKRLEGRSKTWF#(SEQ ID NO:46) ++++ ++ 0.15 181-220$YLLDGMTNTIENARQGAARVTSWLGRQLRIAGKRLEGRSK# (SEQ ID NO:47) ++++ +++ 0.09177-216 $DTALYLLDGMTNTIENARQGAARVTSWLGRQLRIAGKRLE# (SEQ ID NO:48) ++ −0.23 172-211 $GSLLQDTALYLLDGMTNTIENARQGAARVTSWLGRQLRIA# (SEQ ID NO:49) +− >0.33 

Residue Cellular Nuclear Hemolysis number Sequence¹ fluorescencefluorescence (mg/ml) 191-223 $ENARQGAARVTSWLGRQLRIAGKRLEGRSKTWF# +++ +++0.15 (SEQ ID NO:50) 191-220 $ENARQGAARVTSWLGRQLRIAGKRLEGRSK# +++ +++0.31 (SEQ ID NO:51) 191-216 $ENARQGAARVTSWLGRQLRIAGKRLE# ++ + >0.33 (SEQ ID NO:52) 191-211 $ENARQGAARVTSWLGRQLRIA# + − >0.33  (SEQ ID NO:53) 194-220⁴ $RQGAARVTSWLGRQLRIAGKRLEGRSK# ++++ ++++ 0.26 (SEQ ID NO:1)196-220 $GAARVTSWLGRQLRIAGKRLEGRSK# +++ ++ 0.16 (SEQ ID NO:54) 199-220$RVTSWLGRQLRIAGKRLEGRSK# ++ + 0.18 (SEQ ID NO:55) 202-220$SWLGRQLRIAGKRLEGRSK# + + >0.33  (SEQ ID NO:56) 205-220$GRQLRIAGKRLEGRSK# + − >0.33  (SEQ ID NO:25) MAGAININ$GIGKFLHSAGKFGKAFVGEIMKS# − − ? (SEQ ID NO:24) L3 loop$GNGKLIKGRTPIKFGKADCDRPPKHSQNGMGK# ++++ ++++ ? 59-88 (SEQ ID NO:5) ¹$= biotin, #= amide, *= mixture of G and R at that position of thepeptide, bold italic residues differ from E^(ms) of CSFV, strain Alfort.²sequence corresponds to distinct CSFV strains present in sequencedatabase. ³sequence corresponds to BVDV, strain M96751. ⁴sequence ofpeptide showed highest activity.

TABLE 9 Optimization of E^(rns) peptide. Concentration Peptide CodeSequence (μM) Translocation domain E^(rns) MDB-17Biotin-RQGAARVTSWLGRQLRIAGKRLEGRSK-NH2 1.0 according to Table 8 (SEQ IDNO:1) MDL-8 Biotin-RRVTSWLGRQLRIAGKRLEGRSK-NH2 1.0 (SEQ ID NO:57) MDL-9Biotin-RVRSWLGRQLRIAGKRLEGRSK-NH2 1.0 (SEQ ID NO:58) MDA-19Biotin-GRQLRIAGKRLEGRSK-NH2 1.0 (SEQ ID NO:25) MDR-25Biotin-GRQLRIAGKRLRGRSK-NH2 0.3 (SEQ ID NO:59) Optimized transportpeptide MDK-20 Biotin-GRQLRIAGRRLRGRSR-NH2 0.1 (movin) (SEQ ID NO:39)Rhodamine labeled A931 Rhodamine-GRQLRIAGRRLRGRSR-NH2 3.0 (SEQ ID NO:39)Mutations in movin MDM-25 Biotin-GRQLRRAGRRLRGRSR-NH2 0.1 (SEQ ID NO:60)MDM-26 Biotin-GRQLRIAGRRLRRRSR-NH2 0.1 (SEQ ID NO:61) MDM-27Biotin-GRQLRRAGRRLRRRSR-NH2 0.1 (SEQ ID NO:40) Shorter version of movinMDM-28 Biotin-RQLRIAGRRLRGRSR-NH2 0.1 (SEQ ID NO:62) Movin + bromoaceticacid A941 Biotin-RSRGRLRRGAIRLQRG 0.03 biotine with D-aminozuren -Lysine(MTT)- broomacetic acid (retro-inverso) (SEQ ID NO:63)

TABLE 10 Mapping of restrictocin L3 loop. Concentration Peptide Sequence(μM) Restrictocin Biotin-GNGKLIKGRTPIKFGKADCDRPPKHSQNGMGK-NH2 1.0 L3(SEQ ID NO:5) Biotin-GNGKLIKGRTPIKFGKADCDRPPKHSQNGM-NH2 3.0 (SEQ IDNO:64) Biotin-KLIKGRTPIKFGKADCDRPPKHSQNGMGK-NH2 0.3 (SEQ ID NO:65)Biotin-KLIKGRTPIKFGKADCDRPPKHSQNGK-NH2 0.3 (SEQ ID NO:66)Biotin-KGRTPIKFGKADCDRPPKHSQNGMGK-NH2 3.0 (SEQ ID NO:67)Biotin-KLIKGRTPIKFGKADCDRPPKHSGK-NH2 0.3 (SEQ ID NO:68)Biotin-KLIKGRTPIKFGKARCRRPPKHSGK-NH2 0.3 (SEQ ID NO:69)Biotin-KLIKGRTPIKFGK-NH2 (SEQ ID NO:70)

TABLE 11 Translocation activity of transport peptides. ConcentrationName Code Sequence (μM) HRSV-G, type A MDN-12Biotin-KRIPNKKPGKKTTTKPTKKPTIKTTKKDLKPQTTKPK-NH2 1.0 (SEQ ID NO:36)MDN-13 Biotin-KRIPNKKPGKKTTTKPTKKPTIKTTKKDLK-NH2 1.0 (SEQ ID NO:71)MDP-04 Bioton-KRIPNKKPGKKTTTKPTKKPTIKTTKK-NH2 0.3 (SEQ ID NO:72) MDP-08Biotin-KRIPNKKPGKKTTTKPTKKPTIK-NH2 0.3 (SEQ ID NO:73) MDP-19Biotin-KRIPNKKPGKKTTTKPTKK-NH2 0.3 (SEQ ID NO:74) MDP-32Biotin-KRIPNKKPGKKT-NH2  0.03 (SEQ ID NO:42) MDS-34Biotin-KRIPNKKPGKK-NH2  0.03 (SEQ ID NO:9) MDS-36 Biotin-KRIPNKKPKK 0.03 (SEQ ID NO:38) MDS-09 Biotin-KKPGKKTTTKPTKKPTIKTTKK-NH2 0.3 (SEQID NO:75) MDS-23 Biotin-KKPGKKTTTKPTKK-NH2 0.3 (SEQ ID NO:76)Biotin-KKTTTKPTKK-NH2 (SEQ ID NO:77) MDS-37 Biotin-KKPTIKTTKK-NH2 0.3(SEQ ID NO:78) HRSV-G, type B, MDP-21 Biotin-KSICKTIPSNKPKKK-NH2 1.0region 1 (SEQ ID NO:37) MDS-35 Biotin-KTIPSNKPKKK-NH2 0.1 (SEQ ID NO:10)HRSV-G, type B, Biotin-KPRSKNPPKKPK region 2 (SEQ ID NO:11)

Concentration Name Sequence (μM) NLS and DNA/RNA-binding peptides HIV-1Rev Biotin-DTRQARRNRRRRWRERQRAAAAR-NH2 (SEQ ID NO:79) 0.1Biotin-RQARRNRRRRWR-NH2 (SEQ ID NO:30) 0.3/0.1 RSG-1.2Biotin-DRRRRGSRPSGAERRRRRAAAA-NH2 (SEQ ID NO:80) 0.1Biotin-RRRRGSRPSGAERRRRR-NH2 (SEQ ID NO:81) 1.0/0.3 HIV-1 tat/tarBiotin-RPRGTRGKGRRIRR-NH2 (SEQ ID NO:82) 0.3 Bacteriophage lambda NBiotin-QTRRRERRAEKQAQW-NH2 (SEQ ID NO:34) 1.0 peptideBiotin-RRRERRAEK-NH2 (SEQ ID NO:83) 1.0 Flockhouse virus peptideBiotin-NRTRRNRRRVR-NH2 (SEQ ID NO:33)  0.03 Monopartite, NLS simianBiotin-PKKKRKV-NH2 (SEQ ID NO:28) 0.1 virus 40 large T antigen BipartiteBiotin-KRPAAIKKAGQAKKKK-NH2 (SEQ ID NO:29) 0.1 Herpes virus 8 k8 proteinBiotin-TRRSKRRSHRKF-NH2 (SEQ ID NO:32) 0.1 (res 124-135) Proteolyticcleavage site of viral surface protein Alpha virus E3Biotin-KCPSRRPKR-NH2 (SEQ ID NO:35) 3.0 Antibacterial peptideAntibacterial peptide Biotin-RAGLQFPVGRVHRLLRK-NH2 (SEQ ID NO:84) 3.0Buforin

TABLE 12 Peptide concentration that inhibits cell growth. E^(rns)peptide E^(rns) peptide Cell type (191-227) (μM) (194-220) (μM) — HeLa50 40 EBTr 50 60

TABLE 13 Antibacterial effect of E^(rns) or L3 peptides. Peptide¹ MIC(μg/ml)² 191-227 47 191-227 91 194-230 93 184-223 46 181-220 51 177-216224 172-211 >500 191-223 41 191-220 47 191-216 95 191-211 395 194-220 50194-218 25 196-220 25 199-220 23 202-220 179 205-220 >500 L3 >500 ¹Samepeptides as in Table 8. ²Minimal inhibitory concentration needed toinhibit bacterial growth.

1. An isolated peptide domain of 10 to 18 residues long, said isolatedpeptide domain having greater than 40% arginines or lysines and saidisolated peptide domain being at least 70% identical to an amino acidsequence of 10 to 18 residues long located within: about amino acidposition 194 to about amino acid position 220 in a pestiviral E^(rns)protein RNase.
 2. The isolated peptide domain of claim 1, wherein saidpeptide domain is at least 85% identical to said selected amino acidsequence.
 3. An isolated peptide domain of 10 to 18 residues long havinggreater than 40% arginines or lysines and being a reversed amino acidsequence to an amino acid sequence of 10 to 18 residues long locatedwithin: from about amino acid position 194 to about amino acid position220 in a pestiviral E^(rns) protein RNase wherein, further, D-aminoacids are used instead of L-amino acids.
 4. In combination, the isolatedpeptide domain of claim 1 together with a compound for delivery.
 5. Thecombination of claim 4, further comprising means for targeting saidcombination to a specific site.
 6. A pharmaceutical compositioncomprising: an isolated peptide domain of 10 to 18 residues long havinggreater than 40% arginines or lysines and said isolated peptide domainbeing at least 70% identical to an amino acid sequence of 10 to 18residues long located within: about amino acid position 194 to aboutamino acid position 220 in a pestiviral E^(rns) protein RNase; and acompound for delivery.
 7. A method for translocating a compound througha cell's membrane, said method comprising: providing said compound withan isolated peptide domain of 10 to 18 residues long having greater than40% arginines or lysines and said isolated peptide domain being at least70% identical to an amino acid sequence of 10 to 18 residues longlocated within about amino acid position 194 to about amino acidposition 220 in a pestiviral E^(rns) protein RNase; and contacting saidcompound and isolated peptide domain with the cell.
 8. The methodaccording to claim 7, wherein said compound has a molecular weight ofless than about 600 kD.